Description: Electron Paramagnetic Resonance (EPR) spectroscopy is used in many branches of biomedical research. However, the dominant usage in NIH-funded grants is in the field of site-directed spin-labeling (SDSL) using nitroxide-radical spin labels to probe molecular structure. The Technology Research and Development (TR&D) component of this application for continued support of The National Biomedical EPR Center, a P41 Research Resource, focuses on technology enhancement for SDSL applications of EPR. The TR&D component is tightly linked to the Collaborative component, with most collaborations falling in the SDSL classification. Applications involving metal-ions are next among NIH supported grants using EPR, and in this submission special emphasis is placed in the Service component on making EPR capabilities available to this community. Training and Dissemination components are broadly based and vigorous. They include innovation in use of the Internet. Specifically, TR&D Project 1 proposes novel sample-resonator designs. A highlighted sub-aim is development of a new resonator that will increase sensitivity for spin labeled samples in water by a factor of 40 at W-band (94GHz). The increase arises through increase in the amount of sample that can be accommodated from 0.2ul to 25ul. The resonator design concept arose from collaboration with T. Prisner, University of Frankfurt, in developing a dynamic nuclear polarization (DNP) resonator. This Collaboration will continue. Project 2 proposes enhancement of our new W-band bridge, which is configured for convenience in biochemical applications. A highlighted sub-aim is microwave frequency modulation and microwave frequency sweep using a recently developed W-band loop-gap resonator (LGR). Preliminary results are felt to be strong. A collaboration with Dr. A. Beth, Vanderbilt University, will use this capability in a context of Saturation Transfer EPR. Project 3 proposes L-band (1 GHz) for use in distance determination between two spins introduced to a protein by site- specific mutagenesis. The MI = 0 line of perdeutero 14N spin-labels is narrow and very nearly isotropic, making it an ideal probe, but improved instrumentation including an optimized LGR is needed for best sensitivity. The strong support letter for this project from Dr. Hubbell, UCLA, who is also chairman of the Scientific Advisory Board for the Center, is notable. Five of the Collaborations will use this instrumentation, indicating widespread interest in the SDSL community. The Specific Objective of TR&D in this submission is enhancement of EPR instrumentation for SDSL at L-band, where hyperfine interactions dominate, and at W-band where Zeeman interactions dominate. The NIH CRISP database reveals 159 R01 grants that use EPR. We serve the holders of these grants to enhance the nation's biomedical research.